Engineering thioesterase as a driving force for novel itaconate production via its degradation scheme

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
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Abstract

Incorporation of irreversible steps in pathway design enhances the overall thermodynamic favorability and often leads to better bioconversion yield given functional enzymes. Using this concept, here we constructed the first non-natural itaconate biosynthesis pathway driven by thioester hydrolysis. Itaconate is a commercially valuable platform chemical with wide applications in the synthetic polymer industry. Production of itaconate has long relied on the decarboxylation of TCA cycle intermediate cis-aconitate as the only biosynthetic route. Inspired by nature's design of itaconate detoxification, here we engineered a novel itaconate producing pathway orthogonal to native metabolism with no requirement of auxotrophic knock-out. The reversed degradation pathway initiates with pyruvate and acetyl-CoA condensation forming (S)-citramalyl-CoA, followed by its dehydration and isomerization into itaconyl-CoA then hydrolysis into itaconate. Phenylacetyl-CoA thioesterase (PaaI) from Escherichia coli was identified via screening to deliver the highest itaconate formation efficiency when coupled to the reversible activity of citramalate lyase and itaconyl-CoA hydratase. The preference of PaaI towards itaconyl-CoA hydrolysis over acetyl-CoA and (S)-citramalyl-CoA also minimized the inevitable precursor loss due to enzyme promiscuity. With acetate recycling, acetyl-CoA conservation, and condition optimization, we achieved a final itaconate titer of 1 g/L using the thioesterase driven pathway, which is a significant improvement compared to the original degradation pathway based on CoA transferase. This study illustrates the significance of thermodynamic favorability as a design principle in pathway engineering.

工程硫酯酶是通过其降解方案生产新型伊塔康酸的驱动力
在途径设计中加入不可逆步骤可提高整体热力学的有利性,在功能性酶的作用下,往往可获得更高的生物转化产率。利用这一概念,我们在此构建了首个由硫酯水解驱动的非天然衣康酸生物合成途径。衣康酸是一种具有商业价值的平台化学品,在合成聚合物行业有着广泛的应用。长期以来,衣康酸的生产一直依赖于 TCA 循环中间体顺式-乌头酸的脱羧作用,这是唯一的生物合成途径。受大自然中依他康酸解毒设计的启发,我们在此设计了一种与原生代谢正交的新型依他康酸生产途径,无需敲除辅助营养体。这种逆向降解途径以丙酮酸和乙酰-CoA 缩合形成 (S)-citramalyl-CoA 为起点,然后脱水并异构化为 itaconyl-CoA,最后水解为 itaconate。通过筛选确定了大肠杆菌中的苯乙酰-CoA 硫代酯酶(PaaI),当与柠檬醛酸裂解酶和衣康酰-CoA 水合酶的可逆活性结合时,衣康酸的形成效率最高。与乙酰-CoA 和 (S)-citramalyl-CoA 相比,PaaI 更倾向于水解 itaconyl-CoA,这也最大程度地减少了由于酶的杂交性而不可避免的前体损失。通过乙酸酯循环、乙酰-CoA 保护和条件优化,我们利用硫酯酶驱动的途径使伊塔康酸的最终滴度达到了 1 克/升,这与原来基于 CoA 转移酶的降解途径相比有了显著改善。这项研究说明了热力学有利性作为途径工程设计原则的重要性。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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